1. Field of the Invention
The present invention relates, in general, to a host cell and a vector comprising a nucleotide sequence coding for recombinant human etythropoietin (EPO). In particular, the expression vector comprises only one promoter that regulates the EPO expression. The present invention also refers to a method of producing EPO and the EPO thus produced.
2. Background Information
EPO is a glycoprotein that stimulates erythroblast differentiation in the bone marrow, thus increasing the circulating blood erythrocyte count. The mean life of erythrocytes in humans is 120 days and therefore, a human being loses 1/120 erythrocytes each day. This loss must be continuously restored to maintain an adequate level of red blood cells.
The existence of EPO was first postulated by the turn of the century and was definitely proved by Reissman and Erslev early in the ""50s. See Carnot, et al., C.R. Acad. Sci. (France)143:384-386 (1906); Carnot, et al., C.R. Acad. Sci. (France), 143:432-435 (1906); Carnot, et al., C.R. Soc. Biol., 111:344-346 (1906); Carnot, C.R. Soc. Biol., 111:463-465 (1906); Reissman, Blood, 5:372-380 (1950) and Erslev, Blood 8:349-357 (1953). Reissman and Erslev""s experiments were promptly confirmed by other researchers. See Hodgson, et al., Blood, 9:299-309 (1954); Gordon, et al., Proc. Soc. Exp. Biol. Med., 86:255-258 (1954) and Borsook, et al., Blood, 9:734-742 (1954).
The identification of the EPO production site in the organism was an issue of debate. Successive experiments led to the identification of the kidney as the main organ and peritubular interstitial cells as the synthesis site. See Jacobson, et al., Nature, 179:633-634 (1957); Kuratowska, et al., Blood, 18:527-534 (1961); Fisher, Acta Hematol., 26:224-32 (1961); Fisher, et al., Nature, 205:611-612 (1965); Frenkel, et al., Ann. N.Y. Acad. Sci., 149:292-293 (1968); Busuttil, et al., Proc. Soc. Exp. Biol. Med, 137:327-330 (1971); Busuttil, Acta Haematol., (Switzerland), 47:238-242 (1972); Erslev, Blood, 44:77-85 (1974); Kazal, Ann. Clin. Lab. Sci., 5:98-109 (1975); Sherwood, et al., Endocrinology, 99:504-510 (1976); Fisher, Ann. Rev. Pharmacol. Toxicol., 28:101-122 (1988); Jelkmann, et al., Exp. Hematol., 11:581-588 (1983); Kurtz, et al., Proc. Natl. Acad. Sci. (USA), 80:4008-4011 (1983); Caro, et al., J. Lab. Clin. Med., 103:922-931 (1984); Caro, et al., Exp. Hematol., 12:357 (1984); Schuster, et al., Blood, 70:316-318 (1986); Bondurant, et al., Mol. Cell. Biol., 6:2731-2733 (1986); Schuster, et al., Blood, 71:524-527 (1988); Koury, et al., Blood, 71:524-527 (1988); Lacombe, et al., J. Clin. Invest., 81:620-623 (1988); Koury, et al., Blood, 74:645-651 (1989).
A smaller proportion, ranging from 10% to 15% of total EPO, is produced by the liver in adults. See Naughton, et al., J. Surg. Oncol., 12:227-242 (1979); Liu, et al., J. Surg. Oncol., 15:121-132 (1980); Domfest, et al., Ann. Clin. Lab. Sci., 11:37-46 (1981); Dinkelaar, et al., Exp. Hematol., 9:796-803 (1981); Caro, et al., Am. J. Physiol., 244:5 (1983); Dornfest, et al., J. Lab. Clin. Med., 102:274-285 (1983); Naughton, et al., Ann. Clin. Lab. Sci., 13:432-438 (1983); Jacobs, et al., Nature, 313:806-810 (1985); Erslev, et al., Med. Oncol. Tumor. Pharmacother., 3:159-164 (1986). The EPO produced is directly proportional to the extent of tisular hypoxia and its expression rises by increasing the number of the EPO producing cells.
EPO has shown great efficiency in the treatment of anemia, especially anemia derived from renal failure. See Eschbach, et al., N. Enigland J. of Med., 316:73-78 (1987); Krane, Henry Ford Hosp. Med. J., 31:177-181 (1983). Its therapeutical usefulness, however, has been limited due to the unavailability of a massive production method. The quantity and quality of the EPO obtained by the extractive systems known were insufficient. Recently, the use of recombinant DNA technology has made it possible to obtain large amounts of proteins. The application of these techniques to eukaryotic cells has allowed a large-scale production of EPO. See patents U.S. Pat. No. 5,688,679 (to Powell), U.S. Pat. No. 5,547,933 (to Lin), U.S. Pat. No. 5,756,349 (to Lin), U.S. Pat. No. 4,703,008 (to Lin) and U.S. Pat. No. 4,677,195 (to Hewick et al.).
At the present, recombinant DNA techniques are widely known and used. These techniques involve the use of genetic elements such as DNA fragments and enzymes to assemble and transfer genetic constructions for the production of recombinant proteins. The recombinant DNA techniques also facilitate the study of biological mechanisms. See Frank-Kamenetskii, xe2x80x9cUnraveling DNAxe2x80x9d [Samaia Glavnaia Molekula] (Addison Wesley Longman Inc., Reading, Mass., 1997); Brown, xe2x80x9cGene Cloningxe2x80x9d (Chapman and Hall, London, England, 1995); Watson, et al., xe2x80x9cRecombinant DNAxe2x80x9d, 2nd Ed. (Scientific American Books, New York, N.Y., 1992); Alberts et al., xe2x80x9cMolecular Biology of the Cellxe2x80x9d (Garland Publishing Inc., New York, N.Y., 1990); Innis et al., Eds., xe2x80x9cPCR Protocols. A Guide to Methods and Applicationsxe2x80x9d (Academic Press Inc., San Diego, Calif., 1990); Ehrlich, Ed., xe2x80x9cPCR Technology. Principles and Applications for DNA Amplificationxe2x80x9d (Stockton Press, New York, N.Y., 1989); Sambrook et al., xe2x80x9cMolecular Cloning. A Laboratory Manualxe2x80x9d (Cold Spring Harbor Laboratory Press, 1989); Bishop et al., xe2x80x9cNucleic Acid and Protein Sequence. A Practical Approachxe2x80x9d (IRL Press 1987); Reznikoff, Ed., xe2x80x9cMaximizing Gene Expressionxe2x80x9d (Butterworths Publishers, Stoneham, Mass., 1987); Davis et al., xe2x80x9cBasic Methods in Molecular Biologyxe2x80x9d (Elsevier Science Publishing Co., New York, N.Y., 1986); Watson, xe2x80x9cThe Double Helixxe2x80x9d (Penguin Books USA Inc., New York, N.Y., 1969).
The claimed invention comprises an eukaryotic cell line that produces recombinant human EPO, obtained by means of its transfection with an expression vector that comprises a gene coding for human EPO. The vector further comprises an unique promoter and terminator as expression control elements. SEQ ID NO:1 identifies the EPO amino acid sequence codified by the gene used.
The invention provides a host cell comprising a vector which comprises a nucleotide sequence encoding the erythropoietin polypeptide consisting of the amino acid sequence in SEQ ID NO:1, a viral promoter and a viral terminator.
The invention further provides a method for producing an EPO polypeptide, comprising culturing the above host cell under such conditions that said polypeptide is expressed and recovered.
One of the advantages of this invention is that the EPO coding gene utilized does not include non-coding fragments of the 5xe2x80x2 and 3xe2x80x2 regions. However, the system claimed produces an unexpectedly high amount of EPO.
An additional advantage of this invention is the use of expression vectors comprising only one promoter, which exhibit a high EPO productivity. By utilizing the claimed method, it is possible to obtain more than 50 mg of EPO per liter of cell culture per day, that is, over five times the EPO production level claimed by the best method reported so far utilizing one promoter.
The combination of the EPO coding gene claimed in this invention and a simple promoter showed, surprisingly, to operate efficiently, resulting in a stable EPO producing cell. The transfected cells yielded an amount of EPO comparable to, or even higher than, those reported using in theory more adequate, though more complex and difficult to manipulate, genetic constructions.
An additional advantage of the claimed invention is the cotransfection with two vectors that confer different resistance, thus simplifying and facilitating the selection, genetic amplification and maintenance of the cotransfected EPO producing cells.
Further objects and advantages of the present invention will be clear from the description that follows.